EPITHELIAL H+ TRANSPORT--STRUCTURE OF H,K-ATPASE

上皮H运输--H,K-ATP酶的结构

• 批准号: 3244463
• 负责人: ADAM J SMOLKA
• 金额: $ 13.5万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3244463
• 关键词: adenosinetriphosphatase; analytical method; antiulcer drug; apical membrane; autoantibody; chemical structure function; conformation; cytoplasm; electron optics; enzyme structure; epitope mapping; eukaryote; gastric acid; gastric mucosa; gastrointestinal epithelium; gel electrophoresis; high performance liquid chromatography; hydrogen; ion transport; laboratory mouse; laboratory rabbit; membrane transport proteins; microsomes; monoclonal antibody; peptic ulcer; potassium; protein structure function; site directed mutagenesis

The long term goal of this work is to understand the mechanism of H+ secretion by parietal cells of mammalian gastric mucosa. The pathophysiology of gastric ulcers is intimately related to H,K-ATPase activity, which is inhibited directly by omeprazole and indirectly by H2 antagonists. A poorly understood aspect of H+ transport in epithelia is the mechanisms by which the scalar energy of ATP is converted into vectorial energy manifested in a H+ gradient. The H,K-ATPase is a unique experimental model for this mechanism, being readily isolated from the gastric mucosa, where it forms a million fold gradient of H+ across the gastric epithelium. Cloning and sequencing of H,K-ATPase cDNA has revealed the primary structure of the pump. Hydropathy analysis predicts multiple transmembrane alpha-helices which conceivably form H+ and K+ pores. Experimental confirmation or refutation important in modelling these mechanisms is knowledge of H,K-ATPase conformational changes occurring during transport. In this study, the orientation of the H,K-ATPase in the parietal cell apical membrane will be probed with monoclonal antibodies (Mab). The sidedness of Mab and by immunoelectron microscopy. Epitopic domains of the H,K-ATPase. Ligand-induced (E1-E2) and stimulation-induced conformational transitions will be documented using Mab, gastric vesicles, and isolated parietal cells. The study will provide direct evidence for H+ pump topography, will clarify conformational transitions essential to transport, will aid in the interpretation of H,K-ATPase electron diffraction data, will inform site-directed mutagenesis, and will contribute to understanding of the molecular mechanism of H+ transport.

这项工作的长期目标是了解 H+ 的机制 由哺乳动物胃粘膜壁细胞分泌。 这 胃溃疡的病理生理与H,K-ATP酶密切相关 活性，由奥美拉唑直接抑制，由 H2 间接抑制 对手。 上皮细胞中 H+ 转运的一个鲜为人知的方面是 ATP 标量能量转化为能量的机制 矢量能量表现为 H+ 梯度。 H,K-ATP酶是一种独特的 这种机制的实验模型，很容易从 胃粘膜，在胃粘膜上形成百万倍的 H+ 梯度 胃上皮。 H,K-ATPase cDNA 的克隆和测序揭示了 泵的主要结构。 水病分析预测多个 跨膜 α 螺旋可能形成 H+ 和 K+ 孔。 实验证实或反驳对于这些建模很重要 机制是了解发生的 H,K-ATP 酶构象变化 在运输过程中。 在本研究中，H,K-ATP酶在 将用单克隆抗体探测壁细胞顶膜 （马布）。 Mab 的单面性和免疫电子显微镜检查。 表位 H,K-ATP酶的结构域。 配体诱导 (E1-E2) 和刺激诱导 构象转变将使用 Mab、胃囊泡、 和分离的壁细胞。 该研究将为H+提供直接证据 泵拓扑结构，将阐明对于 运输，将有助于解释 H,K-ATPase 电子 衍射数据，将为定点突变提供信息，并将 有助于理解H+传输的分子机制。